The unambiguous identification of proteins/peptides is crucial for the definition of the proteome. Using ProteinChip Array technology (SELDI) we have developed experimental protocols and probed test conditions required for protein identification on ProteinChip Arrays. We are able to directly digest proteins/peptides on-chip surfaces by specific proteases such as trypsin, Glu C and Asp N and obtain the peptide mass fingerprint of the sample under investigation by direct analysis on a simple laser desorption/ionization mass spectrometer. Furthermore, tandem mass spectrometry can be performed on the resulting tryptic peptides by using collision quadrupole time of flight (Qq-TOF) MS/MS via a ProteinChip interface, thus allowing the precise identification of the parent protein within the sample. In addition we are able to identify the C-terminal sequence of peptides after digestion with carboxypeptidase Y directly on ProteinChip surfaces coupled with SELDI-TOF mass spectral analysis both under native and denaturing conditions. Utilizing the on chip digestion methodology, we have examined the structural differences between the disease and physiological forms of proteins that may be diagnostic for the disease processes. The primary structure of both forms of the protein may be identical by MS/MS analysis; however, post-translational modification such as an altered N-linked glycosylation pattern could prove to be informative. By coupling enzymatic digestion(both peptide N-glycosidase F and trypsin) and ProteinChip technology these altered protein forms are identifiable. Furthermore, we have developed methods for studying the interaction of both pathologic and physiologic forms of proteins with their ligands or receptors. The biological characteristics of the pathological form may be altered with regard to their binding properties. Utilizing SELDI, we have also examined the peptide component of patient plasma. In order to fully characterize these molecules we developed a micro-preparative procedure (employing liquid chromatography and SELDI) that provided separation of these peptides. Microsequencing and MS/MS analyses of the peptides have identified several molecules that are fragments of larger proteins that would appear to be different from disease versus control. Combining the resources of the laboratory, we have clearly demonstrated our ability to analyze samples from a variety of disease states, such as Alzheimers disease and Multiple Sclerosis. We are presently evaluating methods for maximizing the number of proteins/peptides detected from CSF. These methods will be used in examining CSF from patients as well as other biological fluids and tissues to identify possible biomarkers for use in earlier diagnosis. By combining both SELDI analysis and on-chip immunoassay, we hope to clearly identify the species that are different from patient versus control as well as those that are post-translationally altered.[unreadable] Currently attempts are being made to ascertain protein interactors in a variety of model systems including the SNARE complex by investigating antibody pull-downs as well as enzymatic cleavage of the complex.